by Poon Hong Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 129-161). / Abstract also in Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT (ENGLISH/CHINESE) --- p.ii / ABBREVIATIONS --- p.vi / LIST OF TABLES --- p.vii / LIST OF FIGURES --- p.viii / TABLE OF CONTENTS --- p.x / INTRODUCTION --- p.1 / LITERATURE REVIEW --- p.4 / Chapter I. --- Models for studying photoreceptor cell death --- p.4 / Chapter II. --- Photic retinopathy --- p.5 / Chapter II.1. --- Proposed Mechanisms of Photic Retinopathy --- p.5 / Chapter II.2. --- Factors Affecting Photic Retinopathy --- p.6 / Chapter II.3. --- Pathologic Processes of Photic Retinopathy --- p.7 / Chapter III. --- P53 --- p.9 / Chapter III.1. --- Historical Perspective --- p.9 / Chapter III.2. --- Structure of p53 --- p.10 / Chapter III.2.1. --- The p53 Gene & mRNA --- p.10 / Chapter III.2.2. --- The p53 Protein --- p.10 / Chapter III.3. --- Modifications of p53 Protein --- p.13 / Chapter III.4. --- Functions of p53 --- p.14 / Chapter III.4.1. --- p53 & Tumors --- p.14 / Chapter III.4.2. --- p53 & DNA Damage --- p.15 / Chapter III.4.3. --- p53 & Apoptosis --- p.16 / Chapter III.4.3.1. --- p53-dependent Apoptosis --- p.16 / Chapter III.4.3.2. --- p53-independent Apoptosis --- p.20 / Chapter IV. --- clusterin --- p.22 / Chapter IV. 1. --- Historical Perspective --- p.22 / Chapter IV.2. --- Structure of Clusterin --- p.22 / Chapter IV.2.1. --- Clusterin Gene & mRNA --- p.22 / Chapter IV.2.2. --- Clusterin Protein --- p.22 / Chapter IV.3. --- Functions of Clusterin --- p.22 / Chapter IV.3.1. --- Clusterin & Apoptosis --- p.23 / OBJECTIVES --- p.24 / MATERIALS AND METHODS --- p.26 / Chapter V. --- Sample collections --- p.26 / Chapter V.l. --- Induction of Photic Injury in Rat --- p.26 / Chapter V.2. --- Tissue Processing --- p.26 / Chapter V.2.1. --- Paraffin-embedded Tissues --- p.26 / Chapter V.2.2. --- Epoxy-embedded Tissues --- p.27 / Chapter V.3. --- Cell Culture --- p.28 / Chapter VI. --- Light-microscopic study --- p.29 / Chapter VI. 1. --- Histopathology --- p.29 / Chapter VI.1.1. --- Toluidine Blue --- p.29 / Chapter VI. 1.2. --- Morphometry of the ONL Thickness --- p.29 / Chapter VI.2. --- In situ TUNEL --- p.29 / Chapter VI.2.1. --- Morphometry of TUNEL --- p.29 / Chapter VI.3. --- Immunohistochemistry --- p.30 / Chapter VI.3.1. --- Single-labeling --- p.30 / Chapter VI.3.1.1. --- Immunolabeling of p53 Protein --- p.31 / Chapter VI.3.1.2. --- Immunolabeling of p21 Protein --- p.31 / Chapter VI.3.1.3. --- Immunolabeling of bax Protein --- p.31 / Chapter VI.3.1.4. --- Immunolabeling of c-fos Protein --- p.32 / Chapter VI.3.1.5 --- Immunolabeling of Clusterin Protein --- p.32 / Chapter VI.3.2. --- Double-labeling --- p.32 / Chapter VI.3.2.1. --- TUNEL & Fluorescent-labeling of p53 --- p.32 / Chapter VI.3.2.2. --- TUNEL & Fluorescent-labeling of p21 --- p.33 / Chapter VI.3.3. --- Grading of Immunoreactivities --- p.33 / Chapter VI.3.4. --- Morphometry of c-fos Immuno-positive Nuclei --- p.33 / Chapter VI.4. --- In situ RT-PCR --- p.34 / Chapter VI.4.1. --- Isolation of Retinal DNA --- p.34 / Chapter VI.4.2. --- Polymerase Chain Reaction (PCR) --- p.34 / Chapter VI.4.3. --- In situ Reverse Transcriptase 226}0ؤ PCR (RT-PCR) --- p.35 / RESULTS --- p.37 / Chapter VII. --- LIGHT-MICROSCOPY --- p.37 / Chapter VII.1. --- Histopathology & Morphometry --- p.37 / Chapter VII. 1.1. --- Histopathology --- p.37 / Chapter VII.1.2. --- Morphometry of the ONL Thickness --- p.49 / Chapter VII.2. --- DNA Nicks Detection --- p.42 / Chapter VII.2.1. --- In situ TUNEL --- p.42 / Chapter VII.2.2. --- Morphometry of TUNEL --- p.42 / Chapter VII.3. --- p53 --- p.51 / Chapter VII.3.1. --- Immunohistochemistry of p53 --- p.51 / Chapter VII.3.2. --- Grading of p53 Immunoreactivities --- p.51 / Chapter VII.3.3. --- p53 in situ RT-PCR --- p.51 / Chapter VII.3.4. --- Double-labeling ofp53 with in situ TUNEL --- p.59 / Chapter VII.4. --- p21 --- p.74 / Chapter VII.4.1. --- Immunohistochemistry of p21 --- p.74 / Chapter VII.4.2. --- Grading of p21 Immunoreactivities --- p.74 / Chapter VII.4.3. --- Double-labeling of p21 with in situ TUNEL --- p.74 / Chapter VII.5. --- bax --- p.84 / Chapter VII.5.1. --- Immunohistochemistry of bax --- p.84 / Chapter VII.5.2. --- Grading of bax Immunoreactivities --- p.84 / Chapter VII.5.3. --- bax in situ RT-PCR --- p.84 / Chapter VII.6. --- c-fos --- p.96 / Chapter VII.6.1. --- Immunohistochemistry of c-fos --- p.96 / Chapter VII.6.2. --- Morphometry of c-fos immuno-positive nuclei & TUNEL --- p.96 / Chapter VII.7. --- Clusterin / Chapter VII.7.1. --- Immunohistochemistry of Clusterin --- p.108 / Chapter VII.7.2. --- Grading of Clusterin Immunoreactivities --- p.108 / discussion --- p.116 / Chapter VIII. 1. --- Summary --- p.116 / Chapter VIII.1.1. --- Histopathology & Morphometry --- p.118 / Chapter VIII.1.2. --- In situ TUNEL --- p.119 / Chapter VIII.1.3. --- p53 & c-fos --- p.119 / Chapter VIII.1.4. --- p27 --- p.124 / Chapter VIII.1.5. --- bax --- p.124 / Chapter VIII. 1.6. --- Clusterin --- p.124 / Chapter VIII.2. --- Hypothesis --- p.125 / Chapter VIII.3. --- Conclusion --- p.127 / bibliography --- p.129

Orientador: Marcelo Carnier Dornelas / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-09-05T12:21:39Z (GMT). No. of bitstreams: 1 Bello_CarolinaCassanoMonte_M.pdf: 4096413 bytes, checksum: 81120541bfee4c3700b982f05823abef (MD5) Previous issue date: 2013 / Resumo: A grande diversidade floral presente entre as espécies de Passiflora é o produto de diversas adaptações à grande variedade de polinizadores. A presença de um verticilo de filamentos denominado corona, entre os verticilos de pétalas e estames, é uma das principais características florais em Passiflora. Estes filamentos são de fundamental importância na interação com polinizadores e suas características (tamanho, forma, cor) estão sob forte pressão de seleção. A coloração dos filamentos da corona pode ter a mesma pigmentação do perianto, ou possuir cores contrastantes. Geralmente esta pigmentação é dada pela presença de antocianinas, garantindo um grande espectro de cores para as flores. Com frequência há uma distribuição diferencial dos pigmentos ao longo dos filamentos da corona. O estudo desta distribuição diferencial dos pigmentos permitiu a identificação de pelo menos 5 tipos de padrões de pigmentação da corona em Passiflora, sendo eles: "dégradée", bandeado e ponteado, bem como pigmentação uniforme e ausência aparente de pigmentação. Devido à grande importância da pigmentação da corona para o sucesso reprodutivo em Passiflora, propôs-se estudar a influência hormonal no processo de pigmentação desta estrutura. Pretendeu-se identificar o efeito da concentração de auxinas (por meio da aplicação de diferentes concentrações de ácido 2,4-diclorofenoxiacético, 2,4-D, e um inibidor do transporte polar de auxina, o ácido naftil-ftalâmico, NPA) e de giberelinas (por meio da aplicação de diferentes concentrações de ácido giberélico, GA3, e de um inibidor de sua síntese, o paclobutrazol, PACLO) na pigmentação de filamentos da corona em desenvolvimento das espécies de P. edulis, P. morifolia e P. suberosa, sendo os tratamentos hormonais realizados in vitro e in planta. Após as aplicações hormonais, os pigmentos florais foram analisados por espectrofotometria UV-vis. A aplicação de GA3 causou aumento da pigmentação floral e a aplicação de um inibidor de sua síntese, PACLO, causou a redução da pigmentação. A Aplicação de auxina (2,4-D) e um inibidor do seu transporte polar (NPA) tiveram efeitos similares no sentido da redução da pigmentação. Determinou-se o padrão de expressão, mediante RT-PCR e hibridização in situ, do gene MYB-R2R3/PACEPS7022E07 de P. suberosa, potencialmente envolvida na modulação da síntese de antocianinas, e demonstrou-se que o mesmo é expresso preferencialmente em órgãos florais / Abstract: The floral diversity present in the species of Passiflora is the product of several adaptations to the wide variety of pollinators. The presence of a whorl of filaments called corona, between the whorls of stamens and petals, is a major floral trait in Passiflora. These filaments are of fundamental importance in the interaction of pollinators and their characteristics (size, shape, color) are under strong selective pressure. The pigmentation of the corona filaments might have the same color of the perianth, or have contrasting colors. Generally the pigmentation is given by the presence of anthocyanins, ensuring a wide range of colors for the flowers. Often there is a differential distribution of pigment throughout the corona filament. Studying a variety of patterns of corona pigmentation, it was established that there are at least 5 types of pigmentation patterns in Passiflora: "dégradée", stripped and dotted, as well as uniformly pigmented and non-pigmented. Due to the great importance of the corona pigmentation for the reproductive success in Passiflora, we proposed to study the influence of hormones on the pigmentation process of this structure. In order to identify the effect of the application of auxins (through different doses of 2,4-dichlorophenoxyacetic acid, 2,4-D, and of the inhibitor of auxin polar transport, naphthyl ftalamic acid, NPA) and gibberellins (through the application of different doses of gibberellic acid, GA3, and its synthesis inhibitor, paclobutrazol, PACLO) in pigmentation of corona filament developing of species P. edulis, P. morifolia and P. suberosa, and hormonal treatments performed in vitro, in cultured corona filaments, and in planta spraying floral buds. For the verification of the effect of hormone applications, the flower pigments were analyzed by spectrophotometer UV-vis where only GA3 caused increased floral pigmentation contrary to PACLO, which caused the reduction of pigmentation. The auxin 2,4-D and an inhibitor of polar auxin transport, NPA had the same effect, reducing the floral pigmentation. We determined the pattern of expression (by RT-PCR and in situ hybridization) of the R2R3-MYB/PACEPS7022E07 gene of P. suberosa, potentially involved in the regulation of anthocyanin synthesis, demonstrating that it is expressed preferentially in floral organs / Mestrado / Biologia Vegetal / Mestra em Biologia Vegetal

A periodontite é uma doença multifatorial, iniciada pela presença de biofilme bacteriano que interage com os mecanismos de defesa do hospedeiro e ocasiona resposta inflamatória quantificável exibindo destruição tecidual. As evidências indicam que a doença periodontal tem estados dinâmicos de exacerbação e remissão. Assim, o objetivo deste estudo foi avaliar a dinâmica da resposta inflamatória ao longo de 45 dias, desde a indução da perda óssea experimental até a crônificação do processo, em modelo de doença periodontal induzido por ligadura. Cinquenta ratos receberam ligadura no 1º molar inferior e foram sacrificados nos seguintes tempos: 1, 3, 5, 7, 10, 15, 20, 30 e 45 dias. Após obtenção das amostras da mandíbula, as peças e as biópsias de gengiva foram processadas para obteção das medidas histomorfométricas e análises microtomográficas e de expressão gênica. Os resultados confirmaram perda óssea progressiva e infiltrado inflamátorio até o tempo de 20 dias (p<0.05). Após 21 dias, observou-se a estabilização da perda óssea. Nos períodos inicias, observou-se predominante expressão de genes pró-inflamatórios, relacionados com a destruição tecidual como: CCL2, CCL3, CCL4, IL1B, IL6, MMP-8, MMP-9 e, nos momentos tardios, uma maior expressão de genes anti-inflamatórios como IL4 e IL10. Através da análise de regressão linear observou-se que a MMP-8 está correlacionada com a expressão de IL1&beta;, TNFa, IL17, MCP1, MIP1a e MIP1b. A MIP1a está correlacionada com MMP2 e RANKL está correlacionada com IL1&beta;, IL17, IL-6, e MIP1b MIP1a. Correlações que mostram uma maior associação a perda óssea nos períodos ativos da doença induzida. Assim, conclui-se que a periodontite experimental, por um período de até 45 dias, passa pelas fazes de exacerbação e remissão. Esse processo dinâmico pode ser monitorado por marcadores moleculares e morfométricos e contribuir para o melhor entendimento da patogênese da doença, contribuindo para a determinação do melhor momento para se avaliar cada biomarcador ou processo biológico. / Periodontitis is a multifactorial disease initiated by the presence of bacterial biofilms hat interacts with the defense mechanisms of the host and causes intense inflammatory response and tissue destruction. Evidence suggests that periodontal disease has dynamic states of exacerbation and remission. Fifty rats received ligation in 1 molar and were sacrificed at the following times: 1, 3, 5, 7, 10, 15, 20, 30 and 45 days, 6 rats per time evaluation. After obtaining the jaw samples, parts and biopsies gum form processed for histomorphometric measures analysis with microtomography and gene expression. Histological results showed microtromograficos and progressive bone loss and inflammation to the influx time of 20 days (p <0.05). After this time it was observed stabilization of bone loss. In the initial periods observed expression of pro-inflammatory genes associated with tissue destruction as CCL2, CCL3, CCL4, IL1B, IL6, MMP8, MMP9 and late times greater expression of anti-inflammatory genes such as IL4 and IL10. Linear regression analysis was used to investigate the possible correlation between levels of MMP expression, FOXP3 and RANKL. MMP8 is correlated with IL1&beta;, TNFa, IL17, MCP1, MIP1a and MIP1b. MIP1a is also correlated with MMP2. RANKL is correlated with IL1&beta;, IL17, IL-6, and MIP1b MIP1a. Correlations that show a greater association with bone loss in active periods of induced disease. Experimental periodontitis, in a period of 45 days, passes through periods of exacerbation and remission. This dynamic process can be monitored by molecular and morphometric markers and contribute to a better understanding of the pathogenesis of the disease, helping to etermine the best time to evaluate each biomarker, or biological process.

A síndrome metabólica (SM) é um conjunto de alterações metabólicas caracterizadas por três de cinco fatores sendo estes; obesidade abdominal, resistência à insulina, hiperglicemia, dislipidemia e hipertensão. As alterações fisiopatológicas da SM são importantes fatores de risco para a doença cardiovascular que tem alta prevalência na maioria das populações. Estudos genéticos têm mostrado que polimorfismos em genes envolvidos em vias do metabolismo glicídico e lipídico estão relacionados com maior predisposição à SM, porém há poucos dados na nossa população. O objetivo deste projeto é estudar a relação entre polimorfismos nos genes LEP, FTO, APOA5, ADRB3, TCF7L2, ENPP1, CYP11B2 e PPARG e a SM. Foram avaliados parâmetros antropométricos, composição corporal, perfil metabólico e de adipocinas, em 167 indivíduos com SM e 262 sem SM, 321 mulheres e 108 homens, e idade entre 30 e 70 anos. Amostras de sangue periférico foram utilizadas para extração de DNA e determinações de perfil lipídico, glicêmico e de adipocinas. Os polimorfismos FTO (rs1421085 T>C, rs1558902 T>A, rs17817449 T>G, rs8050136 C>A, rs9939609 T>A e rs9930506 A>G), LEP rs7799039 G>A, APOA5 rs662799 T>C, ADRB3 rs4994 T>C, TCF7L2 rs7903146 C>T, ENPP1 rs1044498 A>C, CYP11B2 rs1799998 A>G e PPARG rs2972162 C>T foram analisados por PCR em tempo real. As frequências gênicas e alélicas dos polimorfismos estudados foram similares entre os grupos com e sem SM (p>0,05). O haplótipo FTO TTTCAG foi mais frequente no grupo SM (4,2%) do que sem SM (<1,0%, p=0,003), sugerindo que os portadores deste haplótipo tem maior risco de desenvolver síndrome metabólica. Maiores valores de índice de massa corporal (IMC), circunferência abdominal (CA) ou razão cintura-quadril (RCQ) foram observadas nos portadores dos polimorfismos FTO rs1481085 (alelo C), FTO rs1558902 (alelo A), FTO rs17817449 (alelo G), FTO rs8050136 (alelo A), FTO rs9939609 (alelo A), LEP rs7799019 (alelo A), TCF7L2 rs7903146 (alelo C) em comparação com os portadores de genótipos ancestrais (p<0,05), principalmente no grupo SM. Além disso, o polimorfismo FTO rs9930506 (alelo G) foi relacionado com maior teor de gordura corporal (p=0,040), no grupo SM. O SNP CYP11B2 rs1799998 (alelo G) foi relacionado com maior concentração sérica de LDL colesterol e menor concentração de apoAI (p<0,05), grupo SM. Enquanto que a variante ENPP1 rs1044498 (alelo C) foi associada com menor trigliceridemia (p=0,024), no grupo sem SM. Portadores do alelo A do SNP LEP rs7799019 tiveram maior insulinemia e maiores valores de HOMA-&#946; e HOMA-IR que os portadores do genótipo GG (p<0,05), em ambos os grupos, sugerindo a relação desta variante com resistência a insulina. Menores concentrações de leptina foram observadas nos portadores dos SNPs LEP rs7799019 (alelo A) (p=0,031) e ENPP1 rs1044498 (alelo C) (p=0,021) grupo SM; e FTO rs9939609 (alelo A) (p=0,047) no grupo sem SM. A variante FTO rs9930506 (alelo G) foi relacionada com maior adiponectinemia (p<0,05), no grupo SM. Enquanto o SNP ENPP1 rs1044498 (alelo C) foi relacionado com menor concentração sérica de adiponectina nos grupos com (p=0,010) e sem (p=0,006) SM. Em síntese, polimorfismos FTO, LEP e TCF7L2 tem importante papel na adiposidade associada com a síndrome metabólica, em nossa população, e junto com as variantes CYP11B2 ENPP1 contribuem para a variabilidade do perfil metabólico e de adipocinas principalmente em indivíduos com SM. / Metabolic syndrome (MS) is a group of metabolic disorders characterized by three of five factors, which are: abdominal obesity, insulin resistance, hyperglycemia, dyslipidemia and hypertension. The pathophysiological changes of MS are important risk factors for cardiovascular disease that has a high prevalence in most populations. Genetic studies have shown that polymorphisms in genes involved in pathways of glucose and lipid metabolism are associated with increased predisposition to MS, but there are few data on our population. The objective of this project is to study the relationship between polymorphisms in LEP, FTO, APOA5, ADRB3, TCF7L2, ENPP1, PPARG and CYP11B2 and the SM. We evaluated anthropometric parameters, body composition, metabolic profile and adipokines in 167 individuals with MS and 262 without MS, 321 women and 108 men, aged between 30 and 70 years. Peripheral blood samples were used for DNA extraction and determination of lipid profile, glycemic and adipokines. The polymorphisms FTO (rs1421085 T>C rs1558902 T>A rs17817449 T>G rs8050136 C>A rs9939609 T>A and rs9930506 A>G), LEP rs7799039 G>A APOA5 rs662799 T>C ADRB3 rs4994 T>C TCF7L2 rs7903146 C>T, ENPP1 rs1044498 A>C CYP11B2 rs1799998 A>G and PPARG rs2972162C G>T were analyzed by real-time PCR. Genic and allelic frequencies of polymorphisms were similar between the groups with and without MS (p> 0.05). The FTO TTTCAG haplotype was more frequent in the SM group (4.2%) than without MS (<1.0%, p = 0.003), suggesting that carriers of this haplotype have higher risk of developing metabolic syndrome. Higher values of body mass index (BMI), waist circumference (WC) or waist-hip ratio (WHR) were observed in carriers of the FTO rs1481085 polymorphism (C allele), FTO rs1558902 (allele A), FTO rs17817449 (G allele), FTO rs8050136 (allele A), FTO rs9939609 (allele A), LEP rs7799019 (allele A), TCF7L2 rs7903146 (C allele) compared with those with ancestral genotypes (p <0.05), especially in the SM group. In addition, FTO rs9930506 polymorphism (G allele) was associated with higher fat body mass (p = 0.040) in MS group. CYP11B2 SNP rs1799998 (G allele) was associated with increased serum concentration of LDL cholesterol and apoAI lower concentration (p <0.05) SM group. While the ENPP1 variant rs1044498 (C allele) was associated with lower plasma triglycerides (p = 0.024) in the group without MS. Carriers of the A allele of SNP rs7799019 LEP had higher insulin and higher HOMA-&#946; and HOMA-IR than carriers of the GG genotype (p <0.05) in both groups, suggesting the relationship of this variant with insulin resistance. Lower concentrations of leptin were observed in carriers of the LEP SNPs rs7799019 (allele A) (p = 0.031) and ENPP1 rs1044498 (allele C) (p = 0.021) SM group, and FTO rs9939609 (allele A) (p = 0.047) in the group without MS. The variant FTO rs9930506 (G allele) was associated with higher adiponectinemia (p <0.05) in group SM. While the ENPP1 SNPs rs1044498 (C allele) was associated with lower serum concentration of adiponectin in groups with (p = 0.010) and without (p = 0.006) MS. In summary, polymorphisms FTO, TCF7L2 and LEP play an important role in adiposity associated with metabolic syndrome in our population, and along with the CYP11B2 ENPP1 variants contribute to the variability of the metabolic profile of adipokines, especially in individuals with MS.

Cheng Man. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 236-256) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

O aumento do uso de pesticidas para erradicação de pragas e ervas daninhas resultou no aumento da produção agrícola mundial, contudo, levou à preocupação sobre os impactos ambientais, sociais e econômicos decorrentes dessa prática. A microbiota nativa do solo é muito importante para manter a qualidade desse ambiente, mas com o uso intensivo de agroquímicos foram observadas alterações na biomassa microbiana e na formação de grandes quantidades de resíduos tóxicos. O presente estudo teve como objetivo avaliar o potencial genético das frações cultiváveis e não cultiváveis do solo para degradação de três diferentes herbicidas e verificar se a pressão seletiva exercida pela presença de um determinado herbicida tem potencial para alterar a composição da microbiota local, além de identificar os principais gêneros bacterianos cultiváveis da microbiota do solo portadores dos genes de degradação destes herbicidas. Para isso, foram utilizadas seis amostras de solo de diferentes regiões brasileiras, as quais foram obtidas do estado de São Paulo e de áreas de preservação ambiental dos estados do Amazonas e de Goiás. Os principais gêneros bacterianos identificados na microbiota do solo foram: Acinetobacter, Bacillus, Enterobacter, Escherichia, Leclercia, Lysinibacillus, Klebsiella e Staphylococcus e não foram observadas variações nas frações cultiváveis e não cultiváveis do solo quanto ao potencial genético para a degradação dos diferentes herbicidas utilizados.Alguns desses isolados bacterianos apresentaram os genes para a degradação de dois herbicidas (2,4-D e diuron; atrazina e diuron) e ainda isolados que apresentam potencial genético para degradação dos três herbicidas. Além disso, pela análise em HPLC acoplado a espectrometria de massas, algumas bactérias portadoras dos genes de degradação do diuron (puhA e puhB) apresentaram formação dos metabólitos DCPMU, DCPU e DCA, os quais já foram descritos na literatura e também apresentaram metabólitos que ainda não estão descritos na literatura. Por meio das análises de eletroforese em gradiente desnaturante (DGGE) foi possível observar que a pressão seletiva exercida pela presença desses herbicidas altera a composição da microbiota local e a atrazina foi o herbicida que mais afetou a comunidade bacteriana do solo. Portanto, o estudo da diversidade microbiana associada à pressão seletiva causada pelo uso de herbicidas é de grande importância, visto que os herbicidas alteram significativamente a heterogeneidade da comunidade bacteriana do solo. / The use of pesticides to eradicate pests and weeds resulted in increased agricultural production worldwide, however, led to concern about the environmental, social and economic impacts of this practice. Native soil microbiota is very important to maintain the quality of the environment, but with the intensive use of agrochemicals, changes in microbial biomass and formation of large quantities of toxic waste have been observed. This study aimed to evaluate the genetic potential of cultivable and non-cultivable soil fractions to degradate three different herbicides, to verify if the selective pressure exerted by the presence of a particular herbicide has the potential to change the composition of the local microbiota and to identify the main cultivable soil bacterial genera carrying these herbicides degradation genes. For this, six soil samples from different Brazilian regions were used, which were obtained from the state of São Paulo and from environmental preservation areas in the states of Amazonas and Goiás. The main bacterial genera identified in the soil microbiota were: Acinetobacter, Bacillus, Enterobacter, Escherichia, Leclercia, Lysinibacillus, Klebsiella and Staphylococcus, and variations in cultivable and non-cultivable soil fractions genetic potential to degrade various herbicides were observed. Some of these strains harbored genes for degradation of two herbicides (2,4-D and diuron; atrazine and diuron) and other isolates showed genetic potential to degrade one of the three herbicides. Furthermore, analysis with HPLC-MS showed that some bacteria carrying the diuron degradation genes (puhA and puhB) presented formation of metabolites already described in the literature, such DCPMU, DCPU and DCA, and metabolites that have not been described in the literature. By electrophoresis analysis on denaturing gradient (DGGE), was observed that the selective pressure exerted by the presence of these herbicides alters the composition of the local microbiota, being atrazine the herbicide that most affected the bacterial community in the soil. Therefore, the study of the influence of the selective pressure caused by the use of herbicides in the microbial diversity is very important, since herbicides significantly alter the heterogeneity of the soil bacterial community.

Made available in DSpace on 2015-04-11T13:38:48Z (GMT). No. of bitstreams: 1 Nayanne Cristina.pdf: 1826134 bytes, checksum: fa7345995de48191f1f917ea832980fd (MD5) Previous issue date: 2011-06-30 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The production of extended-spectrum beta-lactamases (ESBLs) among clinical isolates of Gram-negative bacteria is an important mechanism of resistance to -lactam antibiotics. It is the major cause of treatment failure using cephalosporins, thereby causing limitations in therapeutic choice and are enzymes mediated by plasmids that have the hability to hidrolisy oximino-cephalosporins and aztreonam. This study aimed to identify key genes responsible for production of ESBLs in Gram-negative Enterobacteriaceae and Pseudomonadaceae family. There were 12 Gram-negative bacteria that were positive for ESBL isolated from patients with hematologic malignancies treated at HEMOAM Foundation from July 2007 to August 2008. The detection and identification of genes blaTEM, blaSHV, blaCTX-M and blaOXA were performed by PCR and nucleotide sequencing. E. coli bacteria were more frequent among ESBL isolates. All isolates were susceptible to imipenem and were resistant to chloramphenicol and tetracycline. The bacteria carried genes for TEM, SHV, CTX-M and OXA on both chromosomes and in plasmids. The presence of multigene (blaTEM, blaSHV, blaCTX-M and blaOXA) and the non-antibiotic resistance detected in the -lactam and non-enterobacteria Enterobacteriaceae isolated from clinical specimens in this research is relevant because it provides information about the presence of this threat antibiotic resistance in our region. / A produção de beta-lactamases de espectro estendido (ESBLs) entre os isolados clínicos de bactérias Gram-negativas é um importante mecanismo de resistência aos antibióticos -lactâmicos. É a maior causa de falha terapêutica utilizando as cefalosporinas, ocasionando assim limitações na escolha terapêutica e são mediadas por plasmídeos e têm a capacidade de hidrolisar oximino-cefalosporinas e aztreonam. Este trabalho teve como objetivo identificar os principais genes responsáveis pela produção das ESBLs em bactérias Gram-negativas da família Enterobacteriaceae e Pseudomonadaceae. Foram estudadas 12 bactérias Gram-negativas que apresentaram fenótipo positivo para ESBL isoladas de pacientes portadores de doenças hematológicas atendidos na Fundação HEMOAM no período de julho de 2007 a agosto de 2008. A detecção e identificação dos genes blaTEM, blaSHV, blaCTX-M e blaOXA foram realizadas por PCR e seqüenciamento nucleotídico. A E. coli foi a bactéria mais freqüente entre os isolados. Todos os isolados ESBL positivos foram suscetíveis ao imipenem e apresentaram resistência ao cloranfenicol e a tetraciclina. As bactérias carreavam genes para TEM, SHV, CTX-M e OXA tanto em cromossomos como em plasmídeos. A presença de multigenes (blaTEM, blaSHV, blaCTX-M e blaOXA) e a resistência a antibióticos não--lactâmicos detectados nas enterobactérias e não-enterobactérias isoladas de amostras clínicas nesta pesquisa se torna relevante pois, oferece informações em nível de saúde pública da presença deste tipo de resistência aos antibióticos em nossa região.

The development of new and better varieties of plants through plant breeding is essential to meet certain needs of a changing world. Genetics and a knowledge of its principles are the basis for such improvement. Barley has been used rather extensively in linkage relations studies. Its desirable characteristics are: (1) interspecific fertility and relative ease of hybridization, (2) numerous characters that are easily differentiated, (3) its commercial importance as a crop and (4) there are seven chromosome pairs in each of the four cultivated species.

Hox genes are regulatory genes encoding small proteins containing a highly conserved 61-amino acid motif, the homeodomain, that enables Hox proteins to bind to DNA at specifically recognised binding sites and transcriptionally activate their target genes. In mammalian species there are 39 Hox genes and they are structural and functional homologs of the Drosophila homeotic complex (Horn-C). During embryogenesis and early development the Hox genes are expressed in a spatiotemporal fashion, where they operate as master transcriptional regulators. Hox genes are further expressed in fully differentiated adult cells, potentially in a tissue-specific manner involving maintenance of the normal phenotype. In selected oncogenic transformations, dysregulated Hox gene expression has been observed, indicating an involvement of these transcriptional regulators in carcinogenesis and metastasis. Utilising quantitative real-time PCR assays, these studies investigated the expression patterns of 20 Hox genes and two wellcharacterised Hox cofactors (Pbx and Meis) in malignant and non-malignant human breast and skin cancer cells. Dysregulated Hox expression was observed for all malignancies tested, of which some misexpressed Hox genes seemed random, whereas other Hox transcripts showed altered levels potentially corresponding with the invasive capacity of the cells. Also, the Hox cofactors Pbx and Meis showed no marked changes in expression levels from the non-malignant to the malignant phenotypes, indicating that it is dysregulated Hox gene expression rather than dysregulated gene expression of Hox cofactors that potentially commit the cell to redifferentiate and undergo oncogenic transformation. Although the Hox proteins are known to be key transcriptional regulators of development, the mechanisms by which they gain their in vivo functional specificity is still largely unknown. They all show strikingly similar transcriptional specificity in vitro, yet show unique specificity in their in vivo environment. This paradox has been the subject of intense scrutiny, however very few direct Hox target genes have been identified, making it a difficult task to decipher the exact manner in which Hox proteins exert their functional potential. Therefore, the studies presented herein were aimed at identifying further Hox target genes in the human system. Utilising differential display approaches, several potential downstream target genes were isolated. Substantiated with real-time PCR assays, one of these potential targets was selected as a likely direct Hox gene target, and as such subjected to further studies. By the combination of bioinformatic analyses, transfection protocols and luciferase assays, a gene encoding the SR-related protein SRrpl3O was shown to be trans-activated in vitro by HOXD4 via a putative Hox binding element within its promoter region. This is the first reported link between Hox transcription factors and the SR and SR-related family of pre-mRNA splicing proteins, offering a new and exciting insight into the complex nature of Hox functional specificity. Finally, this thesis also puts forward new ideas regarding how the Hox proteins gain their transcriptional and functional specificity. Utilising bioinformatic tools in conjunction with performing an extensive review of the disparate catalogue of Hox-related research reports, work herein offers the first comprehensive analysis of the mammalian Hox gene targets in relation to their promoter structures, as well as with respect to the expanded Hox DNA-binding elements. This work reports that identified Hox targets generally contain TATA-less core promoters, many of which have several GC-box elements. The Hox binding elements show no apparent preference regarding their location relative to the transcription start site (TSS), as they are found both upstream and downstream of the TSS, as well as being located close to proximal core promoter elements for some genes and at more distant positions in other gene promoters. Finally, the core Hox binding element TAAT/ATTA contains only part of the necessary recognition sequence involved in Hox-DNA binding, and the notion that flanking base pairs dictate trans-regulatory potential is further explored with the hypothesis that the immediate 3' base pair dictates an activator/repressor-switch of the Hox trans-regulatory effect.

Siglecs and CEA are two families of cell surface proteins belonging to the immunoglobulin superfamily. They are thought to be involved in cell-cell interactions and have various other biological functions. We used the GENECONV program that applies statistical tests to detect gene conversion events in each family of five primate species. For the Siglec family, we found that gene conversions are frequent between CD33rSiglec genes, but are absent between their conserved Siglec genes. For the CEA family, half of gene conversion events detected are located in coding regions. A significant positive correlation was found between the length of the conversions and the similarity of the converted regions only in the Siglec gene family. Moreover, we found an increase in GC-content and similarity in converted regions compared to non-converted regions of the two families. Furthermore, in the two families, gene conversions occur more frequently in the extracellular domains of proteins, and rarely in their transmembrane and cytoplasmic regions. Finally, these two families appear to be evolving neutrally or under negative selection.